Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker

Coupled with over-expression in host organisms, fusion protein systems afford economical methods to obtain large quantities of target proteins in a fast and efficient manner. Some proteases used for these purposes cleave C-terminal to their recognition sequences and do not leave extra amino acids on the target. However, they are often inefficient and are frequently promiscuous, resulting in non-specific cleavages of the target protein. To address these issues, we created a fusion protein system that utilizes a highly efficient enzyme and leaves no residual amino acids on the target protein after removal of the affinity tag. We designed a glutathione S-transferase (GST)-fusion protein vector with a caspase-3 consensus cleavage sequence located between the N-terminal GST tag and a target protein. We show that the enzyme efficiently cleaves the fusion protein without leaving excess amino acids on the target protein. In addition, we used an engineered caspase-3 enzyme that is highly stable, has increased activity relative to the wild-type enzyme, and contains a poly-histidine tag that allows for efficient removal of the enzyme after cleavage of the fusion protein. Although we have developed this system using a GST tag, the system is amenable to any commercially available affinity tag.     

Preparation and functional evaluation of new doxorubicin immunoconjugates containing an acid-sensitive linker on small-cell lung cancer cells

 The anthracycline doxorubicin (DOX) is one of the most effective drugs for the treatment of small-cell lung cancer (SCLC), but its clinical application is limited by unspecific side-effects like cardiotoxicity. In the present study doxorubicin was conjugated to the monoclonal antibodies (mAb) SEN7, MOC31, and SWA11 via a novel acid-sensitive hydrazone linker. These mAb recognize SCLC-associated antigens of cluster 1 (NCAM), cluster 2 (EGP-2/GA733-2), and cluster 4 (CD24) respectively. To assess their potential therapeutic use against SCLC, the antigen-binding activities, the rates of internalization and the cytotoxic effects of the immunoconjugates were examined on tumour cell lines. The preparation procedure preserved the antigen-binding activities of the mAb and yielded immunoconjugates with average drug : mAb ratios of 7 : 1. The hydrazone linker was found to be stable at neutral pH but to release doxorubicin under acidic conditions. In contrast to SEN7-DOX, MOC31-DOX and SWA11-DOX were rapidly internalized into SCLC target cells upon binding to their specific cell-surface antigens. Accordingly, both immunoconjugates proved to be highly cytotoxic agents, inhibiting thymidine incorporation by 50% at concentrations between 0.5 μM and 1 μM and were 100-fold more selective than free doxorubicin. The results suggest that binding to selective cell-surface antigens, rapid internalization and efficient release of doxorubicin from the mAb by acid hydrolysis are required for the selective and potent function of the immunoconjugates. In particular, the use of MOC31-DOX for targeted cytotoxic therapy might be promising because of the limited cross-reactivity of the mAb with normal human tissues and its recently demonstrated tumour localization potential in SCLC patients. Received: 22 September 1995 / Accepted: 23 November 1995     

Perylene attached to DNA through stiff or flexible linker: duplex stability and FRET

Two fluorescent nucleosides, 5-(perylen-3-ylethynyl)-2'-deoxyuridine and 5-[(perylen-3-yl)methoxypropyn-1-yl]-2'-deoxyuridine, were incorporated into synthetic oligodeoxyribonucleotides and spectral properties of the conjugates and their duplexes were studied.     

Novel DNA-directed alkylating agents: Design,synthesis and potent antitumor effect of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker

A series of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker was synthesized for antitumor evaluation. The carbamate or carbonate linker is able to lower the reactivity of the phenyl N-mustard pharmacophore and thus, these conjugates are rather chemically stable. The in vitro studies revealed that these derivatives possessed significant cytotoxicity with IC₅₀ in sub-micromolar range in inhibiting human lymphoblastic leukemia (CCRF-CEM), breast carcinoma (MX-1), colon carcinoma (HCT-116) and human non-small cell lung cancer (H1299) cell growth in vitro. Compounds 10a, 10b, 10e, 10i, and 15a were selected for evaluating their antitumor activity in nude mice bearing MX-1 and HCT-116 xenografts. Remarkably, total tumor remission was achieved by these agents with only one cycle of treatment. Interestingly, no tumor relapse was found in mice treated with 10a over 129 days. This agent is capable of inducing DNA interstrand cross-linking in human non-small lung cancer H1299 cells in a dose dependent manner by modified comet assay and has a long half-life in rat plasma.     

A novel concept for ligand attachment to oligonucleotides via a 2'-succinyl linker

Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects. In this report, a new method for synthesizing oligonucleotide conjugates is described. 2′-Amino-2′-deoxy-5′-dimethoxytrityl-uridine was select ively acylated with a succinic acid linker at the 2′ position. This compound was incorporated at the 3′ end of an oligonucleotide corresponding to the sequence of Oblimersen. The carboxyl group was protected for oligonucleotide synthesis as a benzyl ester, which could be selectively cleaved at the solid phase by a catalytic phase transfer reaction using palladium nanoparticles as catalyst. An oligonucleotide–fluorescein conjugate was prepared by condensation of aminofluorescein. Circular dichroism spectroscopic experiments showed a B-DNA type structure. The melting temperature of the duplex was only slightly lower than that of Oblimersen. Biological activity measured by western blotting resulted in a Bcl-2 target downregulation nearly identical to that of control Oblimersen on human melanoma cells, proving that this method is attractive for the binding of ligands located in the minor groove.     

Rapid calcium release from the isolated sarcoplasmic reticulum is triggered via the attached transverse tubular system

Rapid replacement of 0.15 M K gluconate with 0.15 M choline Cl led to multiphasic Ca2+ release from a heavy fraction of rabbit skeletal muscle microsomes. Following the initial lag period (0-50 ms), about 15 nmol of Ca2+/mg of protein was rapidly released with first-order rate constants k = 60-140 s-1. Subsequently, a larger amount of Ca2+ (up to 56 nmol/mg) was released at a slower rate (k = 0.8-1.5 s-1). The Ca2+ released in both rapid and slow phases was reaccumulated within 60 s. In agreement with a previous report (Caswell, A. H., Lau, Y. H., Garcia, M., and Brunschwig, J-P. (1979) J. Biol. Chem. 254, 202-208), French press treatment of the tubule/sarcoplasmic reticulum (SR) complex results in dissociation of transverse tubular membrane (T-tubules) from SR. Subsequent incubation with 0.4 M potassium cacodylate results in the reassociation of the complex, as shown by sucrose density-gradient sedimentation. Upon T-tubule dissociation, both rapid and slow Ca2+ release was inhibited. Upon reassociation, the rapid Ca2+ release was completely restored and the slow phase partially restored. The results indicate that the T-tubule associated with SR plays a crucial role in triggering rapid Ca2+ release induced by ionic replacement. Other types of Ca2+ release, e.g. those induced by Ca2+ alone or with drugs such as caffeine and quercetin, are unaffected by T-tubule dissociation, and hence produced by direct stimulation of the SR membrane.     

Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1

ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.     

Novel biotin linker with alkyne and amino groups for chemical labelling of a target protein of a bioactive small molecule

We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.     

EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain

The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945–1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.     

EGFP-tagged core and linker histones diffuse via distinct mechanisms within living cells

The effect of chromatin organization on EGFP-tagged histone protein dynamics within the cell nucleus has been probed using fluorescence correlation and recovery measurements on single living HeLa cells. Our studies reveal that free fraction of core-particle histones exist as multimers within the cell nucleus whereas the linker histones exist in monomeric forms. The multimeric state of core histones is found to be invariant across mammalian and polytene chromosomes and this is ATP dependent. In contrast, the dynamics of the linker histones exhibits two distinct diffusion timescales corresponding to its transient binding and unbinding to chromatin governed by the tail domain residues. Under conditions of chromatin condensation induced by apoptosis, the free multimeric fraction of core histones is found to become immobile, while the monomeric linker histone mobility is partially reduced. In addition, we observe differences in nuclear colocalization of linker and core particle histones. These results are validated through Brownian dynamics simulation of core and linker histone mobility. Our findings provide a framework to understand the coupling between the state of chromatin assembly and histone protein dynamics that is central to accessing regulatory sites on the genome.     

Enzymatic glycosylation of reducing oligosaccharides linked to a solid phase or a lipid via a cleavable squarate linker.

Reducing oligosaccharides were converted into their corresponding glycosylamines, and these were reacted with 3,4-diethoxy-3-cyclobuten-1,2-dione (squaric acid diethyl ester). The resulting derivatives could be linked to amino-functionalized lipids, solids, or proteins. Treatment of the obtained lipid or solid conjugates with aqueous bromine or, alternatively, with ammonia-ammonium borate cleaved the linkage and regenerated the oligosaccharide glycosylamines, which were in turn rapidly hydrolyzed to the reducing oligosaccharides. To demonstrate the usefulness of this linkage in enzymatic oligosaccharide synthesis, lactose was linked to a lipid or a solid phase, the obtained conjugates were then subjected to two enzymatic glycosylations (either consecutively or 'one-pot'). The resulting materials were then cleaved to give, in both cases, the expected reducing tetrasaccharide (lacto-N-neotetraose) in good yield.     

Discovery of Novel Inhibitors of a Disintegrin and Metalloprotease 17 (ADAM17) Using Glycosylated and Non-glycosylated Substrates

A disintegrin and metalloprotease (ADAM) proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off-target toxicity. We hypothesized that secondary binding site (exosite) inhibitors should provide a viable alternative to active site inhibitors. Potential exosites in ADAM structures have been reported, but no studies describing substrate features necessary for exosite interactions exist. Analysis of ADAM cognate substrates revealed that glycosylation is often present in the vicinity of the scissile bond. To study whether glycosylation plays a role in modulating ADAM activity, a tumor necrosis factor α (TNFα) substrate with and without a glycan moiety attached was synthesized and characterized. Glycosylation enhanced ADAM8 and -17 activities and decreased ADAM10 activity. Metalloprotease (MMP) activity was unaffected by TNFα substrate glycosylation. High throughput screening assays were developed using glycosylated and non-glycosylated substrate, and positional scanning was conducted. A novel chemotype of ADAM17-selective probes was discovered from the TPIMS library (Houghten, R. A., Pinilla, C., Giulianotti, M. A., Appel, J. R., Dooley, C. T., Nefzi, A., Ostresh, J. M., Yu, Y., Maggiora, G. M., Medina-Franco, J. L., Brunner, D., and Schneider, J. (2008) Strategies for the use of mixture-based synthetic combinatorial libraries. Scaffold ranking, direct testing in vivo, and enhanced deconvolution by computational methods. J. Comb. Chem. 10, 3–19; Pinilla, C., Appel, J. R., Borràs, E., and Houghten, R. A. (2003) Advances in the use of synthetic combinatorial chemistry. Mixture-based libraries. Nat. Med. 9, 118–122) that preferentially inhibited glycosylated substrate hydrolysis and spared ADAM10, MMP-8, and MMP-14. Kinetic studies revealed that ADAM17 inhibition occurred via a non-zinc-binding mechanism. Thus, modulation of proteolysis via glycosylation may be used for identifying novel, potentially exosite binding compounds. The newly described ADAM17 inhibitors represent research tools to investigate the role of ADAM17 in the progression of various diseases.     

Activity of recombinant mitogillin and mitogillin immunoconjugates.

A synthetic gene for the Aspergillus protein toxin mitogillin has been synthesized and expressed in Escherichia coli. The recombinant mitogillin is a potent inhibitor of protein synthesis in vitro with an IC50 of 9.7 pM. Immunoconjugates of recombinant mitogillin derivatized with S-acetylmercaptosuccinic anhydride and 5-methyl-2-iminothiolane modified H65 antibody kill T cell lines and peripheral blood mononuclear cells expressing the human CD5 surface antigen. Native mitogillin contains 4 cysteine residues which form two disulfide pairs (Fernandez-Luna, J. L., Lopez-Otin, C., Soriano, F., and Mendez, E. (1985) Biochemistry 24, 861-867). Three derivatives of mitogillin have been assembled which substitute alanine residues for cysteine residues 5, 147, or 5 and 147. Each of these molecules retains the ability to inhibit protein synthesis in vitro with at most a 2-fold reduction in activity. The derivative mitogillinC147A can be conjugated to 5-methyl-2-iminothiolane- modified H65 antibody directly without pretreatment with S-acetylmercaptosuccinic anhydride, and the immunoconjugate is active against HSB2 cells. Genetic manipulation of toxin genes to expose an accessible cysteine residue into a recombinant product can thus be used to generate immunotoxins without initial derivatization by nonspecific cross-linking reagents.     

The structure of sulfoindocarbocyanine 3 terminally attached to dsDNA via a long, flexible tether

Fluorescence resonance energy transfer (FRET) is an important source of long-range distance information in macromolecules. However, extracting maximum information requires knowledge of fluorophore, donor and acceptor, positions on the macromolecule. We previously determined the structure of the indocarbocyanine fluorophores Cy3 and Cy5 attached to DNA via three-carbon atom tethers, showing that they stacked onto the end of the helix in a manner similar to an additional basepair. Our recent FRET study has suggested that when they are attached via a longer 13-atom tether, these fluorophores are repositioned relative to the terminal basepair by a rotation of ～30°, while remaining stacked. In this study, we have used NMR to extend our structural understanding to the commonly used fluorophore sulfoindocarbocyanine-3 (sCy3) attached to the 5'-terminus of the double-helical DNA via a 13-atom flexible tether (L13). We find that L13-sCy3 remains predominantly stacked onto the end of the duplex, but adopts a significantly different conformation, from that of either Cy3 or Cy5 attached by 3-atom tethers, with the long axes of the fluorophore and the terminal basepair approximately parallel. This result is in close agreement with our FRET data, supporting the contention that FRET data can be used to provide orientational information.     

Analysis of Epstein-Barr virus glycoprotein B functional domains via linker insertion mutagenesis

Epstein-Barr Virus (EBV) glycoprotein B (gB) is essential for viral fusion events with epithelial and B cells. This glycoprotein has been studied extensively in other herpesvirus family members, but functional domains outside of the cytoplasmic tail have not been characterized in EBV gB. In this study, a total of 28 linker insertion mutations were generated throughout the length of gB. In general, the linker insertions did not disrupt intracellular expression and variably altered cell surface expression. Oligomerization was disrupted by insertions located between residues 561 and 620, indicating the location of a potential site of oligomer contacts between EBV gB monomers. In addition, a novel N-glycosylated form of wild-type gB was identified under nonreducing Western blot conditions that likely represents a mature form of the protein. Fusion activity was abolished in all but three variants containing mutations in the N-terminal region (gB30), within the ectodomain (gB421), and in the intracellular C-terminal domain (gB832) of the protein. Fusion activity with variants gB421 and gB832 was comparable to that of the wild type with epithelial and B cells, and only these two mutants, but not gB30, were able to complement gB-null virus and subsequently function in virus entry. The mutant gB30 exhibited a low level of fusion activity with B cells and was unable to complement gB-null virus. The mutations generated here indicate important structural domains, as well as regions important for function in fusion, within EBV gB.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.     

Mimetic ligand-based affinity purification of immune complexes and immunoconjugates

We developed a simple purification method to purify alkaline phosphatase/anti-alkaline phosphatase IgG as immune complexes using mimetic affinity chromatography wherein the antibody was either a monospecific antibody, a bispecific antibody or a commercial polyclonal IgG conjugated with alkaline phosphatase (AP–IgG) covalently. The immune complexes or conjugates were efficiently bound on the mimetic Blue A6XL column and eluted under mild conditions (5–20 mM phosphate buffer). A similar strategy of purifying peroxidase/anti-peroxidase antibody complexes was also successfully demonstrated using the mimetic Red 3 column. Mimetic affinity chromatography thus appears to be a simple method to purify the desired monospecific or bispecific antibodies from the respective hybridomas and quadromas.     

Development of wrapped liposomes: Novel liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids

Novel wrapped liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids were prepared using an efficient, innovative procedure. In this study, dextran fluorescein anionic (DFA) was used as an example of a polyanionic compound. During the process, neutral lipids accumulated around the complexes and eventually covered the complexes. The resulting liposomes were 120-140 nm in diameter and the encapsulation efficiency was up to 90%. In fetal bovine serum, DFA/cationic lipid complexes degraded rapidly but the wrapped liposomes were considerably more stable. Following intravenous administration to rats, DFA/cationic lipid complexes were rapidly eliminated whereas the wrapped liposomes exhibited a much longer blood half-life. These data suggest that DFA is located on the surface of the complexes, but DFA is present inside the wrapped liposomes. The drug-delivery properties of the wrapped liposomes established in the present study suggests that formulations based on this technology could offer important advantages for the administration of many types of drug including antisense oligonucleotides, plasmids and siRNAs which may therefore lead to improved therapeutic effectiveness of this range of drugs. The method of preparation of the wrapped liposomes is so simple that it should be straightforward to adapt to a manufacturing scale.     

Label-free measurement of cell viability via counting cells attached on affinity substrates

The commonly used trypan blue dye exclusion method and other modified cell viability methods, such as fluorescein dye and tetrazolium dye exclusion, artificially introduce toxic chemicals to cells and, thus, alter cellular organelles when measuring cell viability. Therefore, cell viability could be affected by the processes currently used to observe viability. In this study, the cell viability of Chinese hamster ovary (CHO) cells was measured by simply counting attached cells after the cultured CHO cells were attached on a Concanavalin A (Con A) substrate. The efficiency of cell attachment to Con A surfaces was different for live and dead cells allowing the cell viability of CHO cells to be measured without any chemical modifications to the cells.